Human antibodies that inactivate blood coagulation factor VIII fVIII inhibitors, develop in 25 percent of repeatedly transfused patients with severe hemophilia A and rarely in patients with moderate or mild hemophilia. Although the effects of inhibitors are variable, they often contribute to frequent, significant bleeding and disability, and they can present a life threatening complication. In addition, they are responsible for a large proportion of the cost of hemophilia care. The only effective method for inhibitor eradication is high dose fVIII therapy, which decreases inhibitor titers to undetectable levels in about 70 percent of patients, but it is very costly due to the frequent, high fVIII doses required. Patients with low inhibitor titers at the beginning of therapy are more rapidly and successfully tolerized, and tolerance is often long lived (up to 5 yrs.). It is not clear why 30 percent of patients are not tolerized and why it requires longer times in some patients. In 1995, hemophilia A mice were generated by targeted disruption of the murine fVIII gene in exons 16 or 17. We demonstrated that the mice respond to multiple fVIII intravenous injections of doses commonly used in humans by producing anti-fVIII antibodies. Moreover, others showed that splenic T cells from such mice can be stimulated by fVIII to proliferate in vitro. As human fVIII specific T and B cells are difficult to isolate from the peripheral blood, the hemophilic mice present a useful alternative for determining how immunological responses develop and how they can be eliminated. While the fVIII T and B cell epitopes may differ in humans and mice, the fundamental principles of immune recognition and tolerance should be similar. In this proposal we will use the hemophilic mice as a model to test high dose antigen stimulation for tolerance induction. We will determine if additional, simultaneous blocking of T and B cell costimulation by APCs with CTLA4Ig and T-B cell collaboration with anti-CD40L can further downregulate the immune response and whether this is more effective than using high antigen doses alone. Fusion of immunogenic polypeptides to IgG and expression in a retroviral vector introduced into bone marrow cells, leads to tolerance in both the primary and the secondary immune response. This method will also be tested in mice for tolerance induction to fVIII.